Figure - uploaded by Andrew Tolonen
Content may be subject to copyright.
Comparison of glucokinase and galactokinase activities of C. phytofermentans enzymes with those from other species
Source publication
Plant-fermenting Clostridia are anaerobic bacteria that recycle plant matter in soil and promote human health by fermenting dietary fiber in the intestine. Clostridia degrade plant biomass using extracellular enzymes and then uptake the liberated sugars for fermentation. The main sugars in plant biomass are hexoses, and here, we identify how hexose...
Contexts in source publication
Context 1
... bacterium expresses numerous carbohydrate-active enzymes (CAZymes) (5) to degrade plant polysaccharides into hexoses and pentoses of various chain lengths, which are taken into the cell using a panoply of transporters. C. phytofermentans is predicted to carry 572 transporter genes, including 173 genes for sugar transport (6) (see Table S1 in the supplemental material), but the transporters responsible for uptake of the specific saccharides are unknown. ...Context 2
... expression identifies putative hexose transporters. The C. phytofermentans genome is predicted to carry 572 transporter genes by TransportDB (6) and 485 transporter genes by KEGG (17), with 438 genes common to both databases (see Table S1 in the supplemental material). Similar to other bacteria, C. phytofermentans genes encoding a transporter are often cotranscribed as an operon to facilitate their expression at similar levels (18). ...Context 3
... C. phytofermentans, the cphy2241-cphy2243 genes encoding the glucose/galactose ABC transporter are colocated in the genome with a putative hexokinase gene cphy2237, leading us to question whether this enzyme phosphorylates glucose, galactose, or both. We purified Cphy2237 (see Fig. S3 in the supplemental material) and found it phosphorylates galactose (Fig. 6A) with a kinase activity (K m and k cat ) similar to galactokinases from other organisms (Table 1), but this enzyme has undetectable glucokinase activity. Among the six additional genes annotated as glucokinases in C. phytofermentans, we focused on cphy0329 based on its high expression on glucose and homology to the C. beijerinckii glucokinase (21). ...Context 4
... the six additional genes annotated as glucokinases in C. phytofermentans, we focused on cphy0329 based on its high expression on glucose and homology to the C. beijerinckii glucokinase (21). We purified Cphy0329 (Fig. S3) and found it phosphorylates glucose ( Fig. 6B) with an activity (K m and k cat ) similar to other glucokinases (Table 1) but has negligible galactokinase activity. Thus, the glucose/galactose ABC transporter genes are colocated in a gene island with galactokinase, whereas glucose phosphorylation requires an enzyme located elsewhere in the genome that was likely inherited independently. ...Context 5
... subtilis and other bacteria uptake galacturonate using an ExuT transporter in the major facilitator superfamily (MFS) (24,25). C. phytofermentans has no homolog of ExuT, but it does carry 12 putative MFS transporters, 4 of which putatively transport sugars (see Table S1 in the supplemental material). We similarly did not find an individual transporter required for metabolism of cellulose. ...Similar publications
Prostate cancer (PCa) has a high rate of morbidity and mortality, which urges us to find a unique and effective drug for treatment. Brusatol, a triterpenoid-degraded derivative, possesses antitumor activities. However, the significance of Brusatol in prostate cancer has not yet been completely elucidated. Therefore, this study aimed to explore how...
Molecular conformation as a subproblem of the geometrical shaping of the molecules is essential for the expression of biological activity. It is well known that from the series of all possible sugars, those that are most naturally occurring and usable by living organisms as a source of energy—because they can be phosphorylated by hexokinase, the fi...
Mitochondrial metabolites regulate leukaemic and normal stem cells by affecting epigenetic marks. How mitochondrial enzymes localize to the nucleus to control stem cell function is less understood. We discovered that the mitochondrial metabolic enzyme hexokinase 2 (HK2) localizes to the nucleus in leukaemic and normal haematopoietic stem cells. Ove...
Hypoxia is characterized as one of the main consequences of sepsis, which is recognized as the leading cause of death in intensive care unit (ICU) patients. In this study, we aimed to examine whether the expression levels of genes regulated under hypoxia could be utilized as novel biomarkers for sepsis prognosis in ICU patients. Whole blood express...
- [...]
Glucose metabolism is an important metabolic pathway in the auditory system. Chronic alcohol exposure can cause metabolic dysfunction in auditory cells during hearing loss. While alcohol exposure has been linked to hearing loss, the mechanism by which impaired glycolysis promotes cytotoxicity and cell death in auditory cells remains unclear. Here,...
Citations
... These results indicate PTS-dependent processing of the 12 molecules. Although we cannot exclude that other functional elements could be involved in the transport of the substrates tested herein (e.g., hexose permeases or facilitators, which could promote growth on glucose or fructose) [18][19][20] . In addition to the 12 carbon substrates on which growth was abolished, N-acetylmuramic acid yielded diminished growth indicating that this substrate is mainly, but not exclusively, metabolized through PTS. ...
Parageobacillus thermoglucosidasius is a thermophilic bacterium characterized by rapid growth, low nutrient requirements, and amenability to genetic manipulation. These characteristics along with its ability to ferment a broad range of carbohydrates make P. thermoglucosidasius a potential workhorse in whole-cell biocatalysis. The phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) catalyzes the transport and phosphorylation of carbohydrates and sugar derivatives in bacteria, making it important for their physiological characterization. In this study, the role of PTS elements on the catabolism of PTS and non-PTS substrates was investigated for P. thermoglucosidasius DSM 2542. Knockout of the common enzyme I, part of all PTSs, showed that arbutin, cellobiose, fructose, glucose, glycerol, mannitol, mannose, N-acetylglucosamine, N-acetylmuramic acid, sorbitol, salicin, sucrose, and trehalose were PTS-dependent on translocation and coupled to phosphorylation. The role of each putative PTS was investigated and six PTS-deletion variants could not grow on arbutin, mannitol, N-acetylglucosamine, sorbitol, and trehalose as the main carbon source, or showed diminished growth on N-acetylmuramic acid. We concluded that PTS is a pivotal factor in the sugar metabolism of P. thermoglucosidasius and established six PTS variants important for the translocation of specific carbohydrates. This study lays the groundwork for engineering efforts with P. thermoglucosidasius towards efficient utilization of diverse carbon substrates for whole-cell biocatalysis.
... This might be due to plateau zokors' protracted burrowing habits, which maximize their capacity to assist them in perceiving environmental changes in the face of degraded vision. K05685, K10117, and K02027 are all involved in catalytic carbohydrate uptake and directly involved in ATP production (Schneider, 2001;Webb Alexander et al., 2008;Modali and Zgurskaya, 2011;Cerisy et al., 2019). K03406 is involved in bacterial chemotaxis, which is essential for host colonization and virulence of many pathogenic bacteria causing human, animal and plant diseases (Wadhams and Armitage, 2004;Rosenberg et al., 2007;Salah Ud-Din and Roujeinikova, 2017). ...
Gut microbiota not only helps the hosts to perform many key physiological functions such as food digestion, energy harvesting and immune regulation, but also influences host ecology and facilitates adaptation of the host to extreme environments. Plateau zokors epitomize successful physiological adaptation to their living environment in the face of the harsh environment characterized by low temperature, low pressure and hypoxia in the Tibetan plateau region and high concentrations of CO2 in their burrows. Therefore, here we used a metagenomic sequencing approach to explore how gut microbiota contributed to the adaptive evolution of the plateau zokor on the Qinghai-Tibet Plateau. Our metagenomic results show that the gut microbiota of plateau zokors on the Tibetan plateau is not only enriched in a large number of species related to energy metabolism and production of short-chain fatty acids (SCFAs), but also significantly enriched the KO terms that involve carbohydrate uptake pathways, which well address energy uptake in plateau zokors while also reducing inflammatory responses due to low pressure, hypoxia and high CO2 concentrations. There was also a significant enrichment of tripeptidyl-peptidase II (TPPII) associated with antigen processing, apoptosis, DNA damage repair and cell division, which may facilitate the immune response and tissue damage repair in plateau zokors under extreme conditions. These results suggest that these gut microbiota and their metabolites together contribute to the physiological adaptation of plateau zokors, providing new insights into the contribution of the microbiome to the evolution of mammalian adaptation.
... In particular, the increase in Butanoate metabolism and Pyruvate metabolism in the FR group suggested that FR provided more butyrate to participate in energy supply for intestinal epithelial cells . Additionally, enhanced Membrane transport suggested that the synthesis and secretion of metabolites (such as SCFA) and enzymes (such as cellulase) that gut microbiota in the FR group participated in were improved (Cerisy et al., 2019). Previous studies reported that microbiota-derived butyrate augmented the distal gut barrier function via the HIF-1 and AMPK signaling pathway (Kelly et al., 2015;Peng et al., 2009). ...
Based on preparation of co-fermented defatted rice bran (DFRB) using Bacillus subtilis, Saccharomyces cerevisiae, Lactobacillus plantarum and phytase, the present study aimed to evaluate the effects of co-fermented DFRB on growth performance, antioxidant capacity, immune status, gut microbiota and permeability in finishing pigs. Ninety finishing pigs (85.30 ± 0.97 kg) were randomly assigned to 3 treatments (3 replicates/treatment) with a basal diet (Ctrl), a basal diet supplemented with 10% unfermented DFRB (UFR), and a basal diet supplemented with 10% fermented DFRB (FR) for 30 d. Results revealed that the diet supplemented with FR notably (P < 0.05) improved the average daily gain (ADG), gain to feed ratio (G:F) and the digestibility of crude protein, amino acids and dietary fiber of finishing pigs compared with UFR. Additionally, FR supplementation significantly (P < 0.05) increased total antioxidant capacity, the activities of superoxide dismutase and catalase, and decreased the content of malonaldehyde in serum. Furthermore, FR remarkably (P < 0.05) increased serum levels of IgG, anti-inflammatory cytokines (IL-22 and IL-23) and reduced pro-inflammatory cytokines (TNF-α, IL-1β and INF-γ). The decrease of serum diamine oxidase activity and serum D-lactate content in the FR group (P < 0.05) suggested an improvement in intestinal permeability. Supplementation of FR also elevated the content of acetate and butyrate in feces (P < 0.05). Moreover, FR enhanced gut microbial richness and the abundance of fiber-degrading bacteria such as Clostridium butyricum and Lactobacillus amylovorus. Correlation analyses indicated dietary fiber in FR was associated with improvements in immune status, intestinal permeability and the level of butyrate-producing microbe C. butyricum, which was also verified by the in vitro fermentation analysis. These findings provided an experimental and theoretical basis for the application of fermented DFRB in finishing pigs.
... Sugar monomers and oligomers generated extracellularly are imported mainly via ABC (ATP-binding cassette) transport systems in Firmicutes. The genome of M. pectinilyticus (Kim et al., 2019) and multiple additional Firmicutes (Kaoutari et al., 2013;Cerisy et al., 2019) encodes a large number of ABC transporters that are co-localized with CAZyme genes in the genome. Indeed, the role of many of these ABCs for specific sugars uptake has been confirmed by transcriptomic analysis and mutation studies (Cerisy et al., 2019). ...
... The genome of M. pectinilyticus (Kim et al., 2019) and multiple additional Firmicutes (Kaoutari et al., 2013;Cerisy et al., 2019) encodes a large number of ABC transporters that are co-localized with CAZyme genes in the genome. Indeed, the role of many of these ABCs for specific sugars uptake has been confirmed by transcriptomic analysis and mutation studies (Cerisy et al., 2019). This attests to the importance of ABC transport systems for THE uptake of pectin products, as opposed to the alternate sympott/antiport or phosphotransferase (PTS) uptake mechanisms. ...
Pectins are a part of daily diet as well as food additives that are indigestible polysaccharides by human enzymes, however, they can be easily degraded by gut bacteria with the production of short chain fatty acids (SCFAs). Knowledge of pectin gut homeostasis and further how pectin affect gut bacterial communities is insufficient and limited. This review focuses on providing the whole story of how pectin functions as prebiotics in the gut. Understanding the interplay between functional and immunological responses inside animal or human gut as influenced by pectin in diets is provided. The interaction between pectin and gut microbiota is presented from both sides, in terms of how pectin affects gut microbiome and or the fermentation products produced in response by gut bacteria. This knowledge can be used to define preferred dietary pectins, targeting beneficial bacteria, and favoring balanced microbiota communities in the gut to maximize pectins’ health benefits.
... Conjugal transfer of lox71 and lox66 targetron plasmids into C. phytofermentans consistently yielded 10 to 20 transconjugant colonies, of which 80% to 100% contained the expected targetron insertion, indicating that the efficiency of delivery and integration of lox-containing targetrons is similar to that in previous targetrons studies in C. phytofermentans (10,26,27). Following sequential delivery and curing of 2D). ...
... We refer to this double insertion strain as DI-AS (Table 1). Off-site targetron insertions are common (27), which would result in unexpected recombinations. Thus, we applied an inverse PCR assay (27) using primers that anneal to the targetron in order to quantify the number and sequence of genomic targetron insertions. ...
... Off-site targetron insertions are common (27), which would result in unexpected recombinations. Thus, we applied an inverse PCR assay (27) using primers that anneal to the targetron in order to quantify the number and sequence of genomic targetron insertions. This assay confirmed that DI-AS contains both expected genomic targetrons without any additional off-site insertions (Fig. 2E). ...
Clostridia are a group of Gram-positive anaerobic bacteria of medical and industrial importance for which limited genetic methods are available. Here, we demonstrate an approach to make large genomic deletions and insertions in the model Clostridium phytofermentans by combining designed group II introns (targetrons) and Cre recombinase. We apply these methods to delete a 50-gene prophage island by programming targetrons to position markerless lox66 and lox71 sites, which mediate deletion of the intervening 39-kb DNA region using Cre recombinase. Gene expression and growth of the deletion strain showed that the prophage genes contribute to fitness on nonpreferred carbon sources. We also inserted an inducible fluorescent reporter gene into a neutral genomic site by recombination-mediated cassette exchange (RMCE) between genomic and plasmid-based tandem lox sites bearing heterospecific spacers to prevent intracassette recombination. These approaches generally enable facile markerless genome engineering in clostridia to study their genome structure and regulation.
IMPORTANCE Clostridia are anaerobic bacteria with important roles in intestinal and soil microbiomes. The inability to experimentally modify the genomes of clostridia has limited their study and application in biotechnology. Here, we developed a targetron-recombinase system to efficiently make large targeted genomic deletions and insertions using the model Clostridium phytofermentans . We applied this approach to reveal the importance of a prophage to host fitness and introduce an inducible reporter by recombination-mediated cassette exchange.
Control of gene expression is fundamental to cell engineering. Here we demonstrate a set of approaches to tune gene expression in Clostridia using the model Clostridium phytofermentans. Initially, we develop a simple benchtop electroporation method that we use to identify a set of replicating plasmids and resistance markers that can be cotransformed into C. phytofermentans. We define a series of promoters spanning a >100-fold expression range by testing a promoter library driving the expression of a luminescent reporter. By insertion of tet operator sites upstream of the reporter, its expression can be quantitatively altered using the Tet repressor and anhydrotetracycline (aTc). We integrate these methods into an aTc-regulated dCas12a system with which we show in vivo CRISPRi-mediated repression of reporter and fermentation genes in C. phytofermentans. Together, these approaches advance genetic transformation and experimental control of gene expression in Clostridia.
The study of the decomposition of recalcitrant plant biomass is of great interest as the limiting step of terrestrial carbon cycle and to produce plant-derived valuable chemicals and energy. While extracellular cellulose degradation and catabolism have been studied in detail, few publications describe the complete metabolism of hemicelluloses and, to date, the published models are limited to the extracellular degradation and sequential entry of simple sugars.
